Venezuelan equine encephalitis virus (VEE) is a lymphotropic and neurotropic alphavirus, currently causing an explosive human epidemic in Colombia and Venezuela with over 45,000 cases to date. The long-term objectives of the proposed experiments are to define the molecular genetics of VEE induced disease and to use the VEE system to elucidate fundamental aspects of viral pathogenesis. The approach combines molecular genetics with classical histopathology and in situ hybridization. The experiments will utilize a mouse model which approximates VEE disease, molecularly cloned wild-type virus (V3000) which recapitulates the pathogenesis of natural virus isolates; and avirulent or highly attenuated site-directed mutants of V3000. Each of the mutations interdicts the VEE pathogenic sequence at a different stage and therefore links specific phenotypes in vivo with defined mutations. Vector systems have been developed which carry the gene for green fluorescent protein packaged in wild-type or mutant glycoprotein envelopes, facilitating the sensitive detection of ongoing virus replication in living cells or prior replication in preserved tissues. The proposed experiments take advantage of these powerful tools for further genetic dissection of VEE pathogenesis. The experiments are organized into four Specific Aims: 1) To define parameters of the lymphotrophic stage of VEE pathogenesis (e.g. elucidation of the earliest events in infection, identification of lymphoid target cells in vivo and in vitro, and the source of the viremia), 2) To determine the mechanism by which avirulent VEE mutants induce mucosal IgA following subcutaneous inoculation (e.g. characterization of viral spread to inductive sites of the mucosal immune system and correlation of mucosal IgA induction with virus mutation, mouse strain, and ability to spread to inductive sites), 3) To define parameters related to invasion of, dissemination within, and clearance of virus from the CNS (e.g. genetic characterization of CNS invasion, synaptic transmission, persistent infections in cell culture and animals, antibody curing of neuronal infections, and the mechanism of VEE induced neuronal death), and 4) To determine the in vivo and cell culture phenotypes of mutations at nucleotide 3 in the 5' untranslated region of the VEE genome.